Titanium Sulfide Nanosheets Serve as Cascade Bioreactors for H2S‐Mediated Programmed Gas–Sonodynamic Cancer Therapy

Abstract Gas‐mediated sonodynamic therapy (SDT) has the potential to become an effective strategy to improve the therapeutic outcome and survival rate of cancer patients. Herein, titanium sulfide nanosheets (TiS X NSs) are prepared as cascade bioreactors for sequential gas–sonodynamic cancer therapy. TiS X NSs themselves as hydrogen sulfide (H2S) donors can burst release H2S gas. Following H2S generation, TiS X NSs are gradually degraded to become S‐defective and partly oxidized into TiO X on their surface, which endows TiS X NSs with high sonodynamic properties under ultrasound (US) irradiation. In vitro and in vivo experiments show the excellent therapeutic effects of TiS X NSs. In detail, large amounts of H2S gas and reactive oxygen species (ROS) can simultaneously inhibit mitochondrial respiration and ATP synthesis, leading to cancer cell apoptosis. Of note, H2S gas also plays important roles in modulating and activating the immune system to effectively inhibit pulmonary metastasis. Finally, the metabolizable TiS X NSs are excreted out of the body without inducing any significant long‐term toxicity. Collectively, this work establishes a cascade bioreactor of TiS X NSs with satisfactory H2S release ability and excellent ROS generation properties under US irradiation for programmed gas–sonodynamic cancer therapy.


Synthesis of TiS X nanosheets
TiS X nanosheets (TiS X NSs) were synthesized by a high-temperature organic-phase method. Firstly, 20 mL of OM and 10 mL of ODE were mixed in a three-necked flask under vigorous magnetic stirring. The mixture was heated to 120 o C, and then, 440 μL of TiCl 4 was added and maintained at 120 o C for 30 mins with nitrogen protection. Next, the mixture was further heated to 260 o C and 256 mg of S in 4 mL of OM was slowly injected into the solution. Finally, the reaction was maintained at 260 o C for 10 mins under nitrogen protection. After that, the product was naturally cooled down to room temperature and collected by washing with hexamethylene and anhydrous ethyl alcohol.
The TiS X NSs were modified with DSPE-PEG for biomedical applications. Briefly, 20 mg of TiS X NSs and 60 mg of DSPE-PEG were mixed in 6 mL of dichloromethane under ultrasonication for 10 mins, followed by removing dichloromethane through a rotary evaporator. The PEG-TiS X NSs were obtained and re-dispersed in deionized water and stored at 4 o C for future use.

Characterization
The morphologies of TiS X NSs were characterized by transmission electron microscope (TEM, tecnai F20). The crystal structure and surface chemical composition of TiS X NSs were measured by X-ray diffraction (XRD, Panalytical Empyrean) and X-ray photoelectron spectroscopy (XPS, ESCALab 250Xi). The absorption spectra were obtained by UV-vis-NIR spectrophotometer (GenesysTM 10S UV-Vis, Thermo Scientific). Singlet oxygen ( 1 O 2 ) and sulfur vacancy were detected by electron spin resonance (ESR) spectrometer (Bruker EMXplus). The Hainertec (SuZhou) Co., Ltd. Offered the ultrasonic generator. The absolute concentration of Ti ions was measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Avio 200).

Hydrogen sulfide (H 2 S) release of TiS X -PEG NSs
The H 2 S generation was qualitatively analyzed by TMB and MB probes, respectively. Firstly, 200 μL of FeCl 2 (1 mg/mL) and 200 μL of H 2 O 2 (10 mM) were added to 10 mL of deionized water.
Next, 50 μL of TMB (0.5 mM) was added to the above solution to form blue oxTMB by ·OH oxidation. Finally, the PEG-TiS X NSs with different degradation time at various concentrations were added to the above solution, then the declined absorbance of TMB at 655 nm reflected the release of H 2 S by PEG-TiS X NSs, meaning that H 2 S could reverse oxTMB to colorless TMB.
Similarly, the MB probe was with the same principle to reveal the H 2 S generation. Furthermore, H 2 S release was quantitatively analyzed by WSP-1 probe. In short, 50 μL of PEG-TiS X NSs with different concentrations were incubated with 50 μL of WSP-1 probe (50 μM) for 1 h at 37 o C, followed by measuring the fluorescence intensity in Microplate Reader (Ex = 465 nm, Em = 515 nm).

ROS generation of PEG-TiS x NSs by US activation
The DPBF was typically used as a molecular probe to detect the production of ROS. 1 mL of PEG-TiS X NSs (25 μg/mL based on Ti) was mixed with 20 μL of DPBF (1 mg/mL in ethanol solution). The mixture was irradiated with US (30 kHz, 3W/cm 2 ) for different time in the dark. The declined absorbance of DPBF at 420 nm reflected the generation of ROS quantificationally by PEG-TiS X NSs.
To distinguish the types of ROS, TEMP as the trapping agent of 1 O 2 and DMPO as the trapping agent of hydroxyl radical (OH) were used to conduct ESR measurement. 20 μL of TEMP or DMPO was added into 1 mL of PEG-TiS X NSs (15 μg/mL) and irradiated by US (30 kHz, 3 W/cm 2 ) for 2 mins. The ESR spectrometer displayed the characteristic peak of 1 O 2 .

Cellular experiments
The 4T1 murine breast cancer cell line was obtained from American Type Culture Collection (ATCC) and cultured in the standard cell culture medium at the condition of 37 o C, 5% CO 2 . For the cytotoxicity test in vitro, the standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide (MTT) assay was conducted. The different concentrations of PEG-TiS X NSs (0-50 ppm), PEG-D-TiS X NSs (0-100 ppm) were added into the 96-well plates with 4T1 cell, and incubated for various time (6, 12, and 24 h), and the different concentrations of PEG-TiS X NSs (0-50 ppm) were added into the 96-well plates with HUVECs for 12 h. For the study of gas therapy (GT) and sonodynamic therapy (SDT), 4T1 cells were incubated with 25 ppm of PEG-TiS X NSs for 12 h, and then under the US irradiation (30 kHz, 3 W/cm 2 , 1 min per cycle, 5 cycles). The relative cell viabilities were measured by MTT assay.
For the H 2 S detection, 4T1 cells were incubated with the PEG-TiS X NSs in different concentrations for 6 h, and the WSP-1 probe (50 μM) was added to react for 1 h.
For the ATP detection, 4T1 cells were incubated with PEG-TiS X NSs for 12 h. After the different treatments, the cells were collected, and washed three times. Next, the cell members were broken to detect ATP level with the diagnostic kit.

Tumor model
Balb/c mice were purchased from Nanjing Sikerui Biological Technology Co., Ltd, and all the animal experiments were carried out under the permission by Laboratory Animal Center of Soochow University.

The retention and degradation of PEG-TiSx in vivo
The cy5.5 labelled PEG-TiSx NSs (2mg/Kg) were intratumorally (i.t.) injected into the tumor, and the fluorescence signals were observed in the different time. The PEG-TiSx NSs were i.t. injected into the tumor, and the PA signals were detected at 800 nm in the different.

Programmed GT and SDT by cascade bioreactor of PEG-TiSx in vivo
The mice bearing 4T1 tumor (~100 mm 3 ) were randomly divided into eight groups (n=5 per group): (1) Control, (2)  Immune evaluation: When the tumor volumes reached about ~100 mm 3 , the mice bearing 4T1 tumor were randomly divided into two groups and received the following treatments: (1) Control, and (2) TiS X NSs (i.t. injection, 5 mg/kg) (n=5 per group). After the treatments, the mice were sacrificed at the 7 th day. The tumors were homogenized to collect the supernatant solution by the centrifugation. Then, the TNF-α, IL-6, and IL-12-P70 were detected by the enzyme-linked immunosorbent assay.

In vivo toxicity evaluation
For long-term toxicity evaluation, the healthy mice were intravenously injected with PEG-TiS X NSs (10 mg/kg) and sacrificed at different time points (1 st , 7 th , and 14 th , n=5 per time point). The major organs (heart, liver, spleen, lung, kidney, and brain) were collected for both H&E staining, and the Ti ion contents were measured by ICP-OES. Meanwhile, the blood was collected for complete blood panel analysis and blood biochemistry test. To study the metabolism pathway, mice were kept in the metabolic cages to collect the urine and feces after injection of PEG-TiS X NSs at various time points, and the contents of Ti ions were also detected by ICP-OES.

Statistic
All quantitative experiments were done in triplicate unless otherwise indicated. Date are presented as mean ± standard deviation (SD). Statistical differences in survival were measured by the log-rank test. The significance was expressed with *p < 0.05, **p < 0.01, and ***p < 0.001. Figure S1. XRD spectrum of TiS X NSs. Figure S2. XPS spectra of survey of TiS X NSs. Figure S3. The corresponding Raman spectrum of TiS X NSs in Figure 1h.                                                 Figure S54. Blood panel analysis (a-f) and blood biochemistry test (g-i) with healthy Balb/c mice (10 mg/kg). Data were presented as mean values ± SD (n=3 biologically independent mice).